Abstract

Microtubule (MT) plus end-tracking proteins (+TIPs) specifically recognize the ends of growing MTs. +TIPs are involved in diverse cellular processes such as cell division, cell migration, and cell polarity. Although +TIP tracking is important for these processes, the mechanisms underlying plus end specificity of mammalian +TIPs are not completely understood. Cytoplasmic linker protein 170 (CLIP-170), the prototype +TIP, was proposed to bind to MT ends with high affinity, possibly by copolymerization with tubulin, and to dissociate seconds later. However, using fluorescence-based approaches, we show that two +TIPs, CLIP-170 and end-binding protein 3 (EB3), turn over rapidly on MT ends. Diffusion of CLIP-170 and EB3 appears to be rate limiting for their binding to MT plus ends. We also report that the ends of growing MTs contain a surplus of sites to which CLIP-170 binds with relatively low affinity. We propose that the observed loss of fluorescent +TIPs at plus ends does not reflect the behavior of single molecules but is a result of overall structural changes of the MT end.

Analyzing GFP–CLIP-170 on MT ends with FCS. (A) Confocal image of a COS-7 cell transiently transfected with GFP–CLIP-170. The plus sign indicates the location of FCS measurement. Bar, 5 μm. (B) Intensity track of FCS measurement in a transfected COS-7 cell. Peaks of fluorescence are occasionally detected. The dotted red line indicates the exponential fluorescence decay; the bottom double-headed arrows indicate cytoplasmic fluorescence; the top double-headed arrows indicate peak fluorescence. (C) Comparison of the number of cytoplasmic and peak-bound GFP–CLIP-170 particles. Values as depicted in B were measured, and the number of particles was determined for 110 peaks. A scatter plot of these values is best approximated by the curve (indicated by the red line) Y = Ymax × (1 − e−kx).

Fast exchange model. (A) MT polymerization generates a large number of binding sites (orange ellipses), which disappear with single-order reaction kinetics. Thus, as time progresses, less binding sites are present within the depicted rectangle. (B) Dimeric CLIP-170 exchanges rapidly on binding sites irrespective of the position on the MT end. Several interactions with CLIP-170 molecules can occur during the lifetime of a binding site. The equilibrium between cytoplasmic and MT end–bound CLIP-170 (reaction a) might be determined by posttranslational modifications (reaction c), conformational changes, and/or protein–protein interactions. As we find CLIP-170 exchange on MT ends distal of sites of MT polymerization, copolymerization of CLIP-170 with tubulin (reaction b) does not explain the cometlike distribution of +TIPs. However, it is not excluded (hence the stippled arrow), and modified forms of CLIP-170 (indicated by the purple ellipses) might bind tubulin with higher affinity.